Cystic Fibrosis Air3Gel

Product Description

Reproducible and high-throughput in vitro models are essential for studying complex microbial communities. This kit is designed for the in vitro modeling of human disease-specific airway mucus. Cystic Fibrosis Air³Gel is presented here as a platform for culturing microbial samples. The protocol describes how to culture microbial samples in Cystic Fibrosis Air³Gel using a multi-well plate format and how to prepare the samples for downstream analyses. Downstream assays include cell viability assessment, flow cytometry, metabolomics, and sequencing. The potential applications of Cystic Fibrosis Air³Gel include microbial mining, drug screening, and permeability assays.

Components

• 2x multi-well plates containing gradient Cystic Fibrosis Air³Gel.

• 50 mL Bac³Gel® dissolution medium (50 mM sodium citrate).

Reagent Required But Not Provided

• 0.9% (w/v) NaCl solution prepared in deionized water.

Storage Conditions

Store in a dry place inside tightly sealed containers at 2-8 °C.

This product can be stored for at least 9 months.

Precautions and Disclaimer

For R&D use only.

Procedure

Before you begin

• The protocol below describes the specific steps for using single strain samples. However, this protocol has also been used to culture co-cultures, microbial consortia, and complex microbial communities. The Cystic Fibrosis Air³Gel used in this protocol can be produced in Mueller Hinton (MH) medium and is composed of 25.0 mg.mL^-1 porcine stomach type III mucin and 16.3 mg.mL^-1 NaCl. Cystic Fibrosis Air³Gel’s composition is fully tunable; customized compositions are commercially available (i.e. incorporation of other mucin types, proteins, lipids and media). The cross-linking and oxygen gradients within Cystic Fibrosis Air³Gel are intrinsic features established during the production process. These are dynamically affected by microbial culturing.

• Ensure all instruments and consumables (pipettes, falcons, and microcentrifuge tubes) are sterilized and free of contaminants.

• All manipulations should be performed under aseptic conditions, ideally in a Class II biological safety cabinet.

• Prepare a 0.9% (w/v) sterile NaCl solution.

A. Preparation of Microbial Suspensions

Timing: ~ 15 min

1. Grow cultures of the species of interest (e.g. Pseudomonas aeruginosa) to the logarithmic (log) growth phase in the appropriate medium.

• Centrifuge the cultures to pellet the cells, then discard the supernatant.

• Resuspend the cell pellet in an appropriate culture medium.

ImportantImportant

If Cystic Fibrosis Air³Gel was produced in culture medium, researchers should resuspend the cell pellet in 0.9 (w/v) NaCl instead of medium.

• Measure the optical density at 600 nm (OD₆₀₀) of the microbial suspension.

• Adjust the cell concentration as needed.

B. Culturing in Cystic Fibrosis Air³Gel

Timing: ~ 5 min per plate

1. Prepare Cystic Fibrosis Air³Gel multi-well plates at room temperature. For this protocol, 24-well plates are used as example.

2. Add the microbial suspension to each well in a 1:1 volume ratio with Cystic Fibrosis Air³Gel.

Example: For a 24-well plate, add 700 μL of microbial suspension as each well contains 700 μL of Cystic Fibrosis Air³Gel.

Volumes for other plate formats:

• 6-well: 3,700 μL
• 12-well: 1,600 μL
• 48-well: 350 μL
• 96-well: 100 μL

3. Cover the plates and incubate at 37 °C under aerobic or anaerobic conditions for up to 72 hours depending on experimental design.

NoteNote

The recommended incubation period is 72 hours but it may be adjusted as required by the user.

To test the effects of molecules (e.g. biotics, active principles) on microbial samples, it is recommended to let the microbes stabilize in Cystic Fibrosis Air³Gel for 24 hours at 37 °C before adding the molecule of interest.

TipOptional

The multi-well plates can be sealed with a breathable sealing membrane prior to incubation to prevent cross-contamination.

WarningCRITICAL

The microbial suspension should be added gently (to avoid cross-contamination) to the top of Cystic Fibrosis Air³Gel. Do not mix.

While not a strict requirement, ensuring anaerobic conditions prior culturing in Cystic Fibrosis Air³Gel will prevent the loss of obligate anaerobes.

C. Dissolution of Cystic Fibrosis Air³Gel

Timing: ~ 3 min per well

1. For each well, remove the supernatant from the surface of Cystic Fibrosis Air³Gel by gentle aspiration, without disturbing the gel.

• Wash the gels by adding 700 μL (for 24-well plates) of 0.9% NaCl solution.

• Gently aspirate and discard the supernatant.

NoteNote

The washing step ensures that we only consider microbes colonizing Cystic Fibrosis Air³Gel.

2. Add Bac³Gel® dissolution medium (50 mM sodium citrate solution) to each well. Mix thoroughly to fully dissolve Cystic Fibrosis Air³Gel structure.

• To calculate the volume of Bac³Gel® dissolution medium to add to each well, enter the volume (μL) of Cystic Fibrosis Air³Gel according to the multi-well plate typology and click Calculate:

• Transfer the homogenized samples to microcentrifuge tubes.

NoteNote

Full dissolution of Cystic Fibrosis Air³Gel can take some time depending on the plate format and on the presence of exopolysaccharides.

For sequencing: Centrifuge the samples (14,000 x g, 10 minutes), then use the pellet for DNA extraction.

For metabolomics: Centrifuge the samples (14,000 x g, 10 minutes), then collect and lyophilize the supernatant.

Other applications include assessing cell viability, performing flow cytometry analysis, measuring metabolic activity, and conducting standard microbiological characterization assays.

Troubleshooting

Problem 1

The gels are not dissolving properly or take too long to dissolve.

Potential solution

Solution 1: Increase the volume of 50 mM sodium citrate and mix.